Association between SSCPs at Algarvia goat GH and milk traits

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SUMMARY

The DNA from one hundred eight goats be-longing to the indigenous portuguese Algarvia breed was analysed. Single-strand conformation polymorphismÕs (SSCP) were identified at the five exons of the goat Growth Hormone (gGH) gene. Two conformational patterns were found in each of exons 1 and 2, four in exon 3, seven in exon 4 and five in exon 5. The establishment of an asso-ciation of these SSCP patterns with milk, fat and protein production, and fat and protein content was attempted. Patterns A, F and D of exons 2, 4 and 5, respectively, are positively associated with milk, fat and protein production (p<0.05). Pattern A of exon 2 is also positively associated with fat content (p<0.05).

RESUMEN

Se ha analizado el DNA de 108 cabras de la raza aut—ctona Algarvia. Se determinaron los po-limorfismos (SSCPs) en los cinco exones del gen de la GH caprina. Se observaron dos

polimorfis-mos en los exones 1 y 2, cuatro en el ex—n 3, sie-te en el ex—n 4 y cinco en el ex—n 5. Se ha esta-blecido una asociaci—n de estos SSCPs con la producci—n de leche, grasa y prote’na, as’ como con el porcentaje de grasa y prote’na. Los patro-nes A, F e D de los exopatro-nes 2, 4 y 5, respectiva-mente, est‡n positivamente asociados con la producci—n de leche y prote’na (p<0,05). El pa-tr—n A del ex—n 2 est‡ tambiŽn positivamente asociado com el porcentaje en grasa (p<0,05).

INTRODUCTION

Algarvia goat is a Portuguese

indi-genous breed of unknown origin, whose

main potential is milk production that is

used for cheese manufacture. Algarvia

goats are reared mainly in the Algarve

region, where they are very well

adap-ted to dry sylvan areas, and play an

im-portant role as an economic resource to

the rural populations.

A

DDITIONAL KEYWORDS

Polymorphism. PCR-SSCP. Genetic markers.

P

ALABRAS CLAVE ADICIONALES

Polimorfismo. PCR-SSCP. Marcadores genŽticos. ASOCIACIîN ENTRE LOS SSCPs DEL GEN DE LA GH DE LA CABRA ALGARVIA Y

CARACTERêSTICAS DE LA LECHE

Malveiro, E.1_{, P.X. Marques}1_{, I.C. Santos}1_{, C. Belo}2_{and A. Cravador}1

1Unidade de Ci•ncias e Tecnologias Agr‡rias. Universidade do Algarve. Campus de Gambelas. 8000-810 Faro. Portugal. E-mail: acravad@ualg.pt

2Departamento de Ovinicultura e Caprinicultura. Esta•‹o ZootŽcnica Nacional. Quinta da Fonte Boa. 2000-763 Vale de SantarŽm. Portugal. E-mail: ezn.inia@mail.telepac.pt.

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The GH secreted by the pituitary

gland, plays an important role on

lacta-tion. Some GH secretion parameters

and peak frequency are associated with

dairy animals of high genetic value

(Reinecke et al., 1993). It has been

de-monstrated that those animals with high

milk yield reveal superior GH average

levels when compared to those

obser-ved in animals with lower production,

namely during peak lactation

(Bonc-zeck et al., 1988).

Single-strand conformation

poly-morphism (SSCP) is a powerful

me-thod for identifying sequence variation

in amplified DNA. SSCP analysis of

DNA have been used for detection of

genetic mutations in humans (Orita et

al., 1989), rats (Pravenec et al., 1992),

cattle (Kirkpatrick, 1992) and in

va-rious bacteriological (Morohoshi et al.,

1991) and viral (Fujita et al., 1992)

systems. Most significant studies using

the SSCP approach were accomplished

on bovines in linkage analysis

(Nei-bergs et al., 1993) and to define

intra-genic haplotypes at the growth

hormo-ne (Lagziel et al., 1996).

The search for SSCP

polymorphis-mÕs could lead to the finding of genetic

markers useful for improved selection

of agricultural populations, namely

when applied to candidate genes

asso-ciated with quantitative genetic

varia-tion in traits of economic importance.

As part of a programme of genetic

selection aiming at the improvement of

production traits, we have identified

single-strand conformation

polymor-phisms at the five exons of the gGH

gene, by PCR-SSCP analysis. This

work presents preliminary results

to-wards the establishment of an

associa-tion of those polymorphisms with milk,

fat and protein yield, and fat and

pro-tein content.

MATERIAL AND METHODS

DNA samples: DNA was obtained

from peripheral blood leukocytes of

108 animals of Algarvia goat breed

using a DNA Isolation Kit from

Pure-gene.

PCR amplification: The 5 exons

from the gGH gene were amplified by

PCR using the five primer pairs shown

in table I. Five amplification fragments

were generated ranging in size from

112 to 289 bp. PCR reactions were

per-formed in an thermocycler UNOII from

Biometra, using Ready to Go PCR

be-ads, from Amersham Pharmacia

Bio-tech, according to the following

condi-tions: 25 to 50 ng of genomic DNA

(table I); 1.5 U Taq DNA polymerase,

10 mM Tris-HCl, pH 9; 50 mM KCl;

1.5 mM MgCl2; 200 mM of each

dNTP; stabilisers, including BSA, for a

final volume of 25 ml. The

amplifica-tion included 30 cycles of denaturaamplifica-tion

at 95¼C for 30 s, annealing at

57¼C-70¼C (table I) during 30 s and

exten-sion for 30 s at 72¼C. The product of

each amplification was analysed by

electrophoresis on 2 p.100 agarose gel

(5V/cm), using ethidium bromide

stai-ning.

SSCP analysis: For SSCP analysis, 5

ml of each amplification product were

added to 15 ml or 20 ml (table II) of stop

solution (95 p.100 formamide, 10 mM

NaOH, 0.05 p.100 xylene cyanol and

0.05 p.100 bromophenol blue). The

samples were heat-denatured at 95¼C for

5 min, and chilled at 0¼C, and loaded

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Table I. SSCP fragments at gGH gene and PCR analysis parameters.

(Fragmentos de SSCP en el gen de la gGH y an‡lisis de los par‡metros de PCR).

Tannealing Primers DNA Fragment

Exon Length Primer sequence (¡C) (pmol) (ng) localization

(bp)

1 112 5ÕTAA TGG AGG GGA TTT TTG TG3Õ 57 16 25 360-471

5ÕCAG AGA CCA ATT CCA GGA TC3Õ

2 198 5ÕTCT AGG ACA CAT CTC TGG GG3Õ 65 16 50 682-879

5ÕCTC TCC CTA GGG CCC CGG AC3Õ

3 157 5ÕGTG TGT TCT CCC CCC AGG AG3Õ 60 4 25 1063-1219

5ÕCTC GGT CCT AGG TGG CCA CT3Õ

4 200 5ÕGGA AGG GAC CCA ACA ATG CCA3Õ 70 8 25 1416-1615

5ÕCTG CCA GCA GGA CTT GGA GC3Õ

5 289 5ÕAAA GGA CAG TGG GCA CTG GA3Õ 67 16 50 1854-2142

5ÕCCC TTG GCA GGA GCT GGA AG3Õ

Table II. SSCP analysis parameters and results for the indicated exons.

(Par‡metros de an‡lisis por SSCP y resultados para los exones indicados).

DNA/ Vol Run temp Number Frequencies of Exon p.100 T Denat. loades temp patterns patterns

(ml) (¡C) (p. 100) 1 12 1/3 20 15 2 A (97.2 p.100); B (2.8 p.100) 2 10 1/3 20 15 2 A (75.9 p.100); B (24.1 p.100) 3 12 1/3 20 15 4 A (18.5 p.100); B (33.3 p.100;); C (39.8 p.100); D (8.3 p.100) 4 10 1/3 20 15 7 A (13.9 p.100); B (27.8 p.100); C (35.2 p.100); D (5.6 p.100); E (11.1); F (2.8 p.100); G (3.7 p.100) 5 8 1/4 25 20 5 A (14.8 p.100); B (27.8 p.100); C (44.4 p.100); D (2.8 p.100); E (10.2 p.100)

(20 ml or 25 ml of each) onto a 8-12

p.100 polyacrylamide/TBE gel. Gels

were run at 25 W for 4 to 8 hr at 15-20¼C

(table II) in a Dcodeª Universal

Muta-tion DetecMuta-tion System, from BIO-RAD,

coupled with a refrigerated system.

Af-ter the run the gel was removed from the

apparatus and silver stained.

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Table III. Least Square Means (LSMEAN), and Standard Deviation Error (STD Error)

asso-ciated, of milk production parameters for SSCP patterns at exon 1, considering 145 days of

lactation.

(Media de los m’nimos cuadrados (LSMEAN) y respectivo error est‡ndar (STD) de la media de la producci—n de leche para los patrones de SSCP en el ex—n 1, considerando 145 d’as de lactaci—n).

LSMEAN ± STD Error

Pattern Milk (l) Fat content Protein content Fat (kg) Protein (kg)

(p.100) (p.100)

A 115.8 ± 4.68 3.92 ± 0.04 3.94 ± 0.05 4.58 ± 0.21 4.51 ± 0.18 B 150.8 ± 19.35 3.86 ± 0.22 3.84 ± 0.29 5.90 ± 0.86 5.76 ± 0.78

p-value 0.0729 0.7807 0.7191 0.1262 0.1083

Table IV. Least Square Means (LSMEAN), and Standard Deviation Error (STD Error)

asso-ciated, of milk production parameters for SSCP patterns at exon 2, considering 145 days of

lactation.

LSMEAN ± STD Error

(p.100) (p.100)

A 120.8 ± 4.99a 3.97 ± 0.04a 3.98 ± 0.06 4.84 ± 0.22a 4.72 ± 0.19a

B 104.6 ± 7.42b 3.76 ± 0.08b 3.82 ± 0.10 3.92 ± 0.33b 3.96 ± 0.28b

p-value 0.0362 0.0174 0.1563 0.0069 0.0115

a, b - Means with different characters differ statistically (p<0.05)

a, b - Medias con diferentes caracteres difieren estad’sticamente (p<0,05)

Statistical analyses: Statistical

analy-sis was performed on the baanaly-sis of

zoo-technical parameters (milk, fat and

pro-tein production, and fat and propro-tein

con-tent) adjusted to 145 days, related to

1996, 1997 and 1998, as well as

infor-mation about the type of parturition

(one, two or more kids) and animalÕs

age. It was adjusted a mixed linear

mo-del, by the application of SAS PROC

MIXED procedure, to determine

asso-ciations between SSCP and milk

produc-tion factors.

With the following mixed linear

mo-del, we analysed the five exons

separa-tely (because of missing values it was

not possible to perform an analysis for

the five exons simultaneously).

Y

ijk

=

m + tp

i

+

b (x

ijk

- x

_

) +

+

b (x

ijk

- x

_

)

2

_{+ Animal}

_ijk

_{+ Ex}

_j

₊

e

_ijk

Y

_ijk

: observation of the animal k with

parturition type i, pattern level j of exon

m : overall mean

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b (x

ijk

- x

_

): linear effect of age covariate

b (x

ijk

- x

_

)

2

_{: quadratic effect of age}

co-variate

Animal

ijk

: fixed effect

Ex

j

: random effect (exon)

e

ijk

: random error

When the effects were

significati-ve we made use of multiple

compari-son tests (t-test) of LSMEAN values,

Table V. Least Square Means (LSMEAN), and Standard Deviation Error (STD Error)

associa-ted, of milk production parameters for SSCP patterns at exon 3, considering 145 days of

lac-tation.

LSMEAN ± STD Error

(p.100) (p.100) A 119.78 ± 7.91 3.92 ± 0.08 4.03 ± 0.10 4.76 ± 0.35 4.73 ± 0.31 B 116.6 ± 6.49 3.98 ± 0.06 4.05 ± 0.08 4.64 ± 0.29 4.64 ± 0.25 C 117.3 ± 6.38 3.93 ± 0.06 3.86 ± 0.08 4.66 ± 0.28 4.48 ± 0.25 D 108.3 ± 11.30 3.67 ± 0.12 3.67 ± 0.16 3.95 ± 0.50 3.91 ± 0.45 p-value 0.8471 0.1931 0.0993 0.5244 0.4176

Table VI. Least Square Means (LSMEAN), and Standard Deviation Error (STD Error)

asso-ciated, of milk production parameters for SSCP patterns at exon 4, considering 145 days of

lactation.

LSMEAN ± STD Error

(p.100) (p.100) A 128.9 ± 8.56c 4.03 ± 0.10 4.04 ± 0.13 5.26 ± 0.38b 5.08 ± 0.32b B 113.0 ± 6.48bc 4.0 ± 0.07 4.03 ± 0.09 4.54 ± 0.29b 4.51 ± 0.24b C 125.3 ± 6.34c 3.91 ± 0.06 3.91 ± 0.09 4.96 ± 0.28b 4.77 ± 0.24b D 85.7 ± 11.71a 3.67 ± 0.14 3.83 ± 0.18 3.12 ± 0.52a 3.21 ± 0.44a E 110.5 ± 10.26ac 3.87 ± 0.12 3.90 ± 0.16 4.26 ± 0.46ab 4.30 ± 0.39ab F 200.6 ± 21.90d 3.72 ± 0.25 3.52 ± 0.34 7.43 ± 0.98c 6.95 ± 0.83c G 83.3 ± 15.83ab 3.61 ± 0.18 3.72 ± 0.24 2.98 ± 0.71a 3.01 ± 0.60a p-value 0.0001 0.1728 0.6093 0.0002 0.0001

a, b - Means with different characters differ statistically (±p<0.05)

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in an attempt to find the different

le-vel values.

RESULTS AND DISCUSSION

SSCP polymorphisms: We have

chosen exons 1, 2, 3, 4 and 5 of the GH

gene, for the SSCP analysis. The

analy-sis of the amplified fragments by the

PCR method, using the primers

descri-bed in table I is shown in figure 1.

Their lengths correspond to those

ex-pected, according to the position of the

primers, deduced from the described

nucleotide sequence of the gGH gene

from Capra hircus (Kioka et al., 1989).

Considering the one hundred eight

Al-garvia goats, we have observed several

conformational patterns inside each of

those fragments (figure 2 and table II):

two conformational patterns (A and B)

for each of the exons 1 and 2, four in

exon 3 (A, B, C e D), seven in exon 4

(A, B, C, D, E, F e G), and five in exon

5 (A, B, C, D e E). The frequencies

found for each pattern are shown in

ta-ble II.

We have made a preliminary

at-tempt towards the search for

associa-tions between the SSCP

polymor-phisms and quantitative variation in

the productive traits described below.

Tables III, IV, V, VI and VII

summa-rise the results obtained using the

SAS programme applied to the

analy-sis of linear contrasts of SSCPs

rela-ting to milk, protein and fat

produc-tion, and protein and fat content for

each exon analysed. Animals with

pattern A in exon 2 appear to have a

higher milk, fat and protein

produc-tion (p<0.05), and their milk has a

higher fat content than those animals

having pattern B (p<0.05). Animals

with patterns F and D for exons 4 and

Table VII. Least Square Means (LSMEAN), and Standard Deviation Error (STD Error)

asso-ciated, of milk production parameters for SSCP patterns at exon 5, considering 145 days of

lactation.

LSMEAN ± STD Error

(p.100) (p.100) A 121.2 ± 8.43a 3.96 ± 0.10 3.96 ± 0.12 4.84 ± 0.38a 4.74 ± 0.34a B 107.2 ± 6.30a 3.89 ± 0.07 4.01 ± 0.87 4.21 ± 0.28a 4.24 ± 0.25a C 121.2 ± 6.14a 3.97 ± 0.06 3.94 ± 0.08 4.86 ± 0.28a 4.68 ± 0.24a D 201.0 ± 22.43b 3.71 ± 0.25 3.53 ± 0.34 7.45± 1.00b 6.98 ± 0.89b E 118.4 ± 10.25a 3.77 ± 0.12 3.79 ± 0.15 4.49 ± 0.46a 4.43 ± 0.41a P-value 0.0015 0.5109 0.5225 0.0168 0.0390

a, b - Means with different characters differ statistically (±p<0.05)

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5 respectively, are superior milk

pro-ducers (p<0.05).

The SSCP analysis of genes,

who-se product is associated with

produc-tion traits could be a valuable

alterna-tive approach for the establishment of

allelic variants useful as markers to

aid selection. We have applied this

technique to the exons 1, 2, 3, 4 and

5 of GH gene, from the indigenous

Portuguese caprine Algarvia breed.

The SSCP polymorphisms we have

found in the gGH gene coding for a

hormone that exerts a positive

in-fluence in milk production, hint at

the possibility of exploring this

ap-proach for the search of genetic

mar-kers located in this gene. The SSCP

polymorphic variation makes it a

po-tential candidate for the

establis-hment of associations with

quantitati-ve traits. Indeed, statistical analyses

made clear the existence of

associa-tions between the polymorphisms

ob-served in exons 2, 4 and 5 and milk,

protein and fat production, and fat

content. If specific haplotypes can be

defined at this candidate gene that

can be associated with milk

produc-tion, protein and fat content it would

be rendered available a valuable

ge-netic resource for improvement of

this caprine breed.

We aknowledge Funda•‹o para a

Ci•ncia e Tecnologia (FCT) (PRAXIS

XXI 3/3.2/CA/1991/95, INTERREG

18/REGII/6/96) for financial support.

I.C.S. thanks FCT for her PRAXIS

XXI Ph.D. grant (BD/18061/98).

Figure 1. Electrophoretic separation in agarose gel of PCR products (2 p.100 p/v, 5 V/cm)

Lanes 1, 2, 3 - exon 1

Lanes 8, 16 - 100 bp ladder

Lanes 17, 18, 19 - exon 5

Lanes 4, 12 - 50 bp ladder

Lanes 9, 10, 11 - exon 3

Lanes 5, 6, 7 - exon 2

Lanes 13, 14, 15 - exon 4

Separaci—n electroforŽtica en gel de agarosa de los productos del PCR (2 p.100 p/v; 5 V/cm)

Bandas 1, 2, 3 - ex—n 1 Bandas 8, 16 - marcador de 100 pb Bandas 17, 18, 19 - ex—n 5 Bandas 4, 12 - marcador de 50 pb Bandas 9, 10, 11 - ex—n 3

Bandas 5, 6, 7 - ex—n 2 Bandas 13, 14, 15 - ex—n 4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 300 bp 200 bp 100 bp 50 bp Ð +

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Figure 2. Left: SSCPÕs patterns observed in non denaturant PAGE; middle: schematic

repre-sentation of GH gene with exons represented by black boxes. Numbers represent the

localisa-tion of amplified fragments; right: schematic representalocalisa-tion of SSCP patterns for each exon.

(Izquierda: patrones SSCP observados en PAGE no desnaturalizante; centro: representaci—n esquem‡tica del gen GH con exones representados por los cuadros negros. Los nœmeros representan la localizaci—n de los fragmentos amplificados; derecha: representaci—n esquem‡tica de los patrones SSCP para cada ex—n). Exon 360 471 Exon 682 879 Exon 1063 1219 Exon 1416 1615 Exon 1854 2142 2544 1 A B A B A B C D A B C D E F A B C D E B A B A A B B B B A A C C D C A B C D E F A A B B C C D D

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